1. 1 Department of Biology and CESAM, University of Aveiro, 3810 Aveiro, Portugal 2 Medinfar– Pharmaceutical Products SA, Amadora, 2700 Venda Nova, Portugal 3 Institut für Chemie, Technische Universität Berlin, 10623 Berlin, Germany Tânia Caetano 1,2, Joanna Krawczyk 3, Eva Mösker 3, Roderich D. Süssmuth 3, Sónia Mendo 1 Lichenicidin is a class II two-component lantibiotic produced by B. licheniformis. It is composed of the two peptides Bli  and Bli , which act synergistically against various Gram- positive bacteria, including MRSA and Listeria monocytogenes. The lichenicidin gene cluster was successfully expressed in Escherichia coli, constituting the first report of a complete lantibiotic gene cluster heterologous expression in a Gram-negative host. This system was further exploited to characterize and assign the function of proteins encoded in the biosynthetic gene cluster in the maturation of lichenicidin peptides. Abstract Heterologous expression, biosynthesis and mutagenesis of type II lantibiotic from Bacillus licheniformis in Escherichia coli References 1) Dischinger, J., Josten, M., Szekat, C., Sahl, HG., Bierbaum, G. PLoS One. 2009, 4, e6788. 2) Datsenko, K.A., and Wanner, B.L. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 6640-6645. 3) Gust, B., Challis, G.L., Fowler, K., Kieser, T., and Chater, K.F. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 1541-1546. 4) Begley, M., Cotter, PD., Hill, C., Ross, RP. Appl Environ Microbiol. 2009, 75, 5451-5460. 5) Shenkarev, ZO., Finkina, EI., Nurmukhamedova, EK., Balandin, SV., Mineev. KS., Nadezhdin, KD., Yakimenko, ZA., Tagaev, AA., Temirov, YV., Arseniev, AS., Ovchinnikova, TV. Biochemistry. 2010, 49, 6462-6472. Analysis of mutants Bioassay of lichenicidin gene inactivation mutants Mutagenesis of the lichenicidin biosynthetic cluster ABAB PCR + pLic5ΔX pLic5 pKD20 pLic5ΔX E. coli EPI300 pLic5ΔX pIJ778 upstream primer downstream primer disruption cassettte E.coli BW25113 AB C DE BmtI Funding Fig. 4: Agar diffusion assay for the assessment of lichenicidin production by knockout mutants of the lic gene cluster with M. luteus as the indicator strain. (A) Antibacterial activity exhibited by each of the mutants constructed in this study. (B) Synergetic activity of peptides Bliα and Bli , produced by Lic5ΔA2 and Lic5ΔA1, respectively. (C) Restored lichenicidin activity was observed when Bli  and Bliα peptides could interact. Mutants Lic5ΔA1 and Lic5ΔA2 are exclusive producers of Bli  and Bliα, respectively. Purify genomic DNA Randomly shear & end-repaid DNA Isolate DNA of correct size Perform in-gel ligation Screen The lichenicidin gene cluster Fig. 3: λ-RED recombinase system [2, 3] was used to carry out the inactivation of all lic genes except those related with immunity. A) Amplification of the disruption cassette B) PCR-targeting. The fosmid pLic5 (containing the lichenicidin biosynthetic gene cluster) was introduced into BW25113 cells containing the RED recombinase expression plasmid pKD20 [2]. C) Isolation of pLicΔX D) Restriction digestion of pLicΔX with BmtI and the plasmid religation E) Transformation of E. coli EPI300 with pLicΔX. Construction and screening of a fosmid library of B. licheniformis Fig. 1: (A) Representation of lichenicidin biosynthetic gene cluster organization [1], according to the genome annotation for Bacillus licheniformis ATCC 14760. Black circles correspond to deduced Rho-dependent terminators (B) Proposed structures of Bliα and Bliβ peptides [4, 5]. Fig. 2: A fosmid library of B. licheniformis I89 was screened for the presence of the lichenicidin gene cluster by colony blot hybridization with lanA1/A2 DIG labeled probe. Positive colonies were submitted to PCR and the amplified fragment sequenced. Fig. 5: ESI-mass spectra of Bliα (3249.54 Da) and Bli  (3019.38 Da) peptides detected in B. licheniformis I89 supernatant (A) and cell (B) extracts. Genetic inactivation of the structural genes licA1 and LicA2 resulted in the production of only Bli  (C) and Bliα (D), respectively.